Fibrinogen is a dimeric protein, each half of which is composed of disulfide-bonded polypeptide chains designated Aα, Bβ and γ. In the liver, the genes for the Aα- and Bβ-chains encode single products of 610 and 461 amino acid residues, respectively. In contrast, alternative splicing of transcripts of the γ-chain gene yield γ-chain variants of slightly different lengths (411 and 427 residues), the shorter of which constitutes ˜90% of the final product. The predominant form of fibrinogen is secreted into the circulation with a molecular mass of ˜340 kDa. Following its secretion from the liver, the protein exists not only in plasma, but also in lymph and interstitial fluid. In healthy individuals, the concentration of fibrinogen in plasma is between 4 and 10 μM. Importantly, that concentration can increase by as much as much as 400% during times of physiological stress.
Fibrinogen is converted to fibrin by thrombin, a trypsin-like serine proteinase. Thrombin hydrolyzes at least two specific Arg-Gly bonds within fibrinogen. This process leads initially to the formation of fibrin protofibrils which can associate laterally, forming thicker fibres that, in turn, can associate to form even thicker and branched fibrin bundles. Such bundles are further stabilised by cross-links formed between Lys and Gln residues located within the α-chains of neighboring fibrin molecules, to form a 3-D meshwork capable of preventing or limiting blood flow. This process of cross-linking is catalysed by the enzyme Factor XIIIa.
Two types of congenital abnormalities of fibrinogen exist, afibrinogenemia and dysfibrinogenemia. Afibrinogenemia is a quantitative deficiency that results in bleeding diatheses. The term hypofibrinogenemia refers to a less severe fibrinogen deficiency. Dysfibrinogenemia is marked by functional abnormalities of fibrinogen that may result in either bleeding or thrombosis. Patients may be treated with fibrinogen concentrate or cryoprecipitate. Fibrinogen is also commonly used during surgery as an adjunct to hemostasis in the form of fibrin sealants. These are typically two-component systems comprising fibrinogen (e.g. in the form of fibrinogen concentrate) and thrombin in appropriate pharmaceutical compositions.
A concern in the administration of these partially purified forms of fibrinogen is the presence of contaminating proteins. An affinity-based purification method for the isolation of fibrinogen would therefore be useful. Such a purification method would be especially useful if it were able to isolate fibrinogen from non-depleted plasma, in a specific fashion, leaving all other protein components intact for further manipulation.
WO97/10887 discloses triazine-based compounds, useful as affinity adsorbents, of formula I
wherein R1 is H, alkyl, hydroxyalkyl, cyclohexyl, NH2, phenyl, naphthyl, 1-phenylpyrazole, indazole, benzthiazole, benzoxazole or benzimidazole, any of which aromatic groups can be substituted with one or more of alkyl, alkoxy, acyloxy, acylamino, amino, NH2, OH, CO2H, sulphonyl, carbamoyl, sulphamoyl, alkylsulphonyl and halogen;
one X is N and the other is N, C—Cl or C—CN;
Y is O, S or NR2;
Z is O, S or NR3;
R2 and R3 are each H, alkyl, hydroxyalkyl, benzyl or β-phenylethyl;
Q is benzene, naphthalene, benzthiazole, benzoxazole, 1-phenylpyrazole, indazole or benzimidazole;
R4, R5 and R6 are each H, OH, alkyl, alkoxy, amino, NH2, acyloxy, acylamino, CO2H, sulphonic acid, carbamoyl, sulphamoyl, alkylsulphonyl or halogen;
n is 0 to 6;
p is 0 to 20; and
A is a support matrix, optionally linked to the triazine ring by a spacer.
Compounds of formula I are disclosed as having affinity for proteins such as immunoglobulins, insulin, Factor VII or human growth hormone.
Compounds of related structure are disclosed in WO00/67900 and WO03/097112. They have affinity for endotoxins.